The higher efficiency of A-T cloning compared with blunt-end ligation has been shown before. In addition, the Y-adapters are ligated in a sticky-end fashion, with a nontemplated A-overhang at the 3′-ends of the DNA fragments in combination with a T-overhang of the adapters. After ligation of this adapter to both fragment ends, both DNA strands are available for sequencing, yielding a higher proportion of functional molecules (up to 100% of the DNA strands). The advantage of these Y-shaped adapters is that one adapter comprises the necessary functionalities for both the 5′- and the 3′-ends. By contrast, generic sequencing on Illumina platforms is based on so-called Y-adapters. In addition, only 25% of the total input DNA strands are sequenceable overall, since only one strand of the functional fragments (A-P1) will be clonally amplified. As a result, the sequence information stored in the other half of ligated fragments carrying identical adapters on both ends (A-A or P1-P1) is lost. In these A-P1 libraries, where A and P1 represent the two different linear adapters, statistically only 50% of the ligated sequence fragments carry the combination of A and P1 adapter that is obligatory for sequencing (A-P1).
THE ICE SEQUENCE EPUB DOWNLOAD TORRENT TORRENT
In a generic Ion Torrent library preparation workflow, two different linear adapters (A and P1) are ligated onto the DNA fragments in order to incorporate the necessary functionalities for sequencing. In both cases, each library fragment needs to have specific sequence features at the 5′- and the 3′-ends, respectively, in order to be functional ( Figure 1). Nevertheless, they share some characteristics of the shotgun DNA library preparation process: the necessity of DNA fragmentation to a defined fragment size range followed by fragment end polishing and the addition of platform-specific adapters by ligation. Both platforms differ in the two key features of clonal library amplification and sequence detection. Today, Illumina and Ion Torrent instrument families are dominating the market of second-generation sequencing platforms. Therefore, efficient conversion of genetic material into a sequenceable library is urgently necessary. Library preparation represents a decisive step in a sequencing workflow, and problems or limitations at this point can jeopardize the whole sequencing project as the available input material may be limited. High-throughput sequencing is widely used for a variety of sequence-based analyses in different fields.
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